Angiotensin II type 1 receptor localizes at the blood–bile barrier in humans and pigs

Animal models and clinical studies suggest an influence of angiotensin II (AngII) on the pathogenesis of liver diseases via the renin–angiotensin system. AngII application increases portal blood pressure, reduces bile flow, and increases permeability of liver tight junctions. Establishing the subcellular localization of angiotensin II receptor type 1 (AT1R), the main AngII receptor, helps to understand the effects of AngII on the liver. We localized AT1R in situ in human and porcine liver and porcine gallbladder by immunohistochemistry. In order to do so, we characterized commercial anti-AT1R antibodies regarding their capability to recognize heterologous human AT1R in immunocytochemistry and on western blots, and to detect AT1R using overlap studies and AT1R-specific blocking peptides. In hepatocytes and canals of Hering, AT1R displayed a tram-track-like distribution, while in cholangiocytes AT1R appeared in a honeycomb-like pattern; i.e., in liver epithelia, AT1R showed an equivalent distribution to that in the apical junctional network, which seals bile canaliculi and bile ducts along the blood–bile barrier. In intrahepatic blood vessels, AT1R was most prominent in the tunica media. We confirmed AT1R localization in situ to the plasma membrane domain, particularly between tight and adherens junctions in both human and porcine hepatocytes, cholangiocytes, and gallbladder epithelial cells using different anti-AT1R antibodies. Localization of AT1R at the junctional complex could explain previously reported AngII effects and predestines AT1R as a transmitter of tight junction permeability. Supplementary Information The online version contains supplementary material available at 10.1007/s00418-022-02087-z.


Supplementary Methods
Plasmid construction. The human wild-type full-length AT1R cDNA (accession No. NM_000685; cDNA Resource Center (www.cdna.org), CloneID: AGTR100000, catalogue no. AGTR10TN01) was subcloned into a modified episomal expression vector pCEP-Pu (kindly provided by Prof. Dr. Manuel Koch, Center for Biochemistry and Institute for Dental Research and Oral Musculoskeletal Biology, University of Cologne), after introduction of the restriction sites NheI and XhoI. The same restriction sites were used for cloning. The vector backbone contained a carboxyl-terminal thrombin cleavage site and a double Strep-tag® II sequence inframe with N-terminal BM-40 signal peptide (Gara et al. 2008). The construct pCEP-Pu-AGTR1 was confirmed by sequencing (Eurofins Genomics Germany GmbH).
Puromycin selection (3μg/mL) was started 24h post transfection. Cells were kept under continuous selection pressure and split when 80-90% confluent.
For immunocytochemistry, cells expressing heterologous human AT1R (hhAT1R) or control cells were seeded on glass coverslips and cultured in selection medium. Coverslips were carefully removed when cells were 60-80% confluent, 3h air-dried at RT, and stored at -20°C.
Pre-adsorption with blocking peptide. For pre-adsorption studies, a commercial blocking peptide (sc-31181P, Lot C2814, Santa Cruz Biotechnology Inc., USA) was used according to the manufacturer's instructions with an antibody to blocking peptide ratio of 1:1 (w/w).

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Cryosections of human and porcine liver were incubated with anti-AT1R-C18 antibody with or without pre-adsorption with blocking peptide. Specimens of hhAT1R-expressing HEK293-EBNA cells were similarly incubated side-by-side either with anti-AT1R-C18 or anti-AT1R-G3 antibodies or using the same antibodies pre-adsorbed with blocking peptide. The sequence of the blocking peptide was identified by mass spectrometry (Central Bioanalytics, CMMC, Cologne, Germany).
Preparation of whole cells lysates and plasma membrane. Whole cell lysates were obtained by a modified method according to Sharma et al. (Sharma et al. 2012). In brief, 80-90% confluent hhAT1R-expressing HEK293-EBNA or control cells were mechanically harvested and immediately transferred into conical tubes. Cells were centrifuged (200RCF, 5min at 4°C) and cell pellets were suspended in ice cold PBS. After repeating the washing step twice, the resulting pellets were lysed in ice cold lysis buffer (10mM Tris, 1mM EDTA, 1% SDS, 0.1% Triton X-100, and 1mM phenylmethylsulfonyl fluoride, 200µl/10 6 cells) supplemented with cOmplete (Roche) and stored on ice for 60min. Subsequently, the lysate was centrifuged at 20,800RCF for 60min at 4°C and the supernatants were aliquoted, immediately frozen and stored at -80°C until use. For enrichment of the plasma membrane the method of Lund et al. was employed (Lund et al. 2009). Briefly, the cells were mechanically harvested as before.
Finally, cell pellets were suspended in 3mL HB, incubated for 5min on ice, and centrifuged for 5min at 310RCF, 4°C. Cell pellets were suspended in 1.5mL lysis buffer (255mM sucrose, 20mM HEPES pH 7.4, 1mM EDTA supplemented with cOmplete (Roche)) and 3min homogenized on ice using a motor-driven potter (500rpm). The homogenates were centrifuged for 10min at 20,080RCF at 4°C to remove cell debris. Supernatants were transferred into S5 ultracentrifuge tubes and centrifuged for 200min in a TLA-55 rotor (Beckman) at 54,000rpm at 4°C. Supernatants were carefully removed and pellets were dissolved in 50µL PBS including cOmplete (Roche), frozen in liquid N2 and stored at -20°C. Tissue samples were directly homogenized in 1× Laemmli buffer containing cOmplete protease inhibitor cocktail (Roche).
Protein concentration of the samples was estimated by Bradford using BSA as standard.
SDS-PAGE and Western blotting. Proteins were mixed with 2× Laemmli sample buffer containing 1% SDS and 50mM dithiothreitol. Alternatively, the membrane pellets were homogenized in 6M urea, 1% SDS, and 10mM TCEP. In both cases, reaction mixtures were incubated for 10min at RT. Increasing protein amounts were separated on 10% SDS-acrylamide gels. After electrophoresis, the proteins were transferred onto nitrocellulose-membranes, 0.2µm (#88024, Thermo ScientificTM) using Tobwin transfer buffer (192mM glycine, 25mM Tris, 0.01% SDS, and 10% methanol). Blots were blocked for 30min at RT with 5% (w/v) skimmed milk powder in PBS containing 0.1% Tween-20 and incubated overnight at 4°C with primary antibody. The following day, blots were incubated with the appropriate horseradish peroxidaseconjugated secondary antibodies for 1h. Protein bands were visualized with PierceTM ECL Western Blotting Substrate (#32106, Thermo ScientificTM) by exposure of the immunoblots to Amersham HyperfilmTM ECL (#28906835, GE Healthcare, Germany). Quantification of protein bands was performed by densitometric scans using the software Phoretix 1D (Version 5.00) with Rubber Band algorithm for background subtraction (Biostep, Germany).

Results and Discussion
Effect of treatment and fixatives of liver cryo-sections on recognition of AT1R by anti-AT1R-C18 antibody. In air-dried, Triton-X100-permeabilized, untreated or acetone-treated cryosections of fresh-frozen, i. e. unfixed liver tissue, anti-AT1R-C18 antibody detected AT1R in plasma membranes of hepatocytes with a tram-track-like appearance ( Supplementary Fig.   S6 1a). Treatment with methanol or methanol/acetone (1:1) produced a fuzzy signal non-related to morphological structures, probably due to loss of epitope recognition. Aldehyde fixation of the cryosections for 10 minutes at room temperature with 2% in PBS prior to incubation with anti-AT1RC18 antibody resulted in an aggregation-like appearance of the signals with scarcely visible tram-track-like patterns. Increasing the PFA concentration to 4% caused further aggregation of anti-AT1R-C18 with concomitant reduction of tram-track-associated signal.
As expected (Mardones and Gonzalez 2003), in air-dried, Triton-X100-permeabilized cryosections of unfixed tissue that were otherwise untreated prior to immunocytochemical incubation, no redistribution of membrane proteins took place, but the drawback was a low preservation of morphological structures. Acetone, as organic solvent and coagulative fixative, removed lipids, caused cytoplasmic flocculation, and tissue shrinkage. The latter is a wellknown problem of most fixatives (Tran et al. 2015;Noguchi et al. 1997). However, acetone instantaneously stabilizes the cell membrane and precipitates proteins to their cellular architecture by dehydration (Hughes and Jones 2011;Hall et al. 1987;Bhattacharyya et al. 2010;Horobin 1982). Consequently, acetone precipitated AT1R with its seven transmembranespanning domains to the plasma membrane and retained it in the cell compartment without redistribution. Whereas acetone did not preserve morphological structures as PFA does, it preserved a plethora of epitopes that are destroyed by PFA fixation (Noguchi et al. 1997). In sections fixed with 2% PFA, anti-AT1R-C18 was able to recognize mainly membraneassociated epitopes and revealed only seldom track-like structures. Loss of immunoreactivity after PFA fixation is attributed to formation of inter-and intramolecular methylene bridges between the amino groups of proteins (Scalia et al. 2017). PFA fixation has been reported to induce blebs in plasma membrane (Fox et al. 1985), and hence is responsible for morphological artifacts. We therefore refrained from using tissues fixed with PFA or methanol, as no antigenantibody interactions or insufficient interactions were observed.

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Specificity of secondary antibodies and autofluorescence. For evaluation of the antibodies in human and porcine liver sections, we determined the specificity of the secondary fluorescent antibodies and autofluorescence of the investigated specimens. This is exemplary shown for human and porcine liver cryosections and secondary anti-goat Alexa®568 antibody in Supplementary Fig. 1b, 11a and 11d. With exception of the internal elastic membrane of arteries ( Supplementary Fig. 3a), neither specific secondary antibody signals nor autofluorescence were observed under the chosen settings.
Detection of AT1R by anti-AT1R antibodies. Previous studies found several antibodies from commercial sources to be unspecific and giving false positive signals (Herrera et al. 2013a;Benicky et al. 2012;Rateri et al. 2011;Herrera et al. 2013b;Michel et al. 2009;Bouressam et al. 2018). Due to the ongoing debate concerning the specificity of anti-AT1R antibodies, we first singled out antibodies that were capable of specifically detecting AT1R by immunohistochemistry (IHC) and immunocytochemistry (ICC) (Supplementary Fig. 7-12) (Lorincz and Nusser 2008;Fritschy 2008). Initially, we employed six anti-AT1R antibodies to corroborate the localization of AT1R (Supplementary Table 1). With these antibodies we aimed not only to detect the C-terminus of AT1R, but also the N-terminus and/or the central region of the protein. However, plasma membrane-associated AT1R localization was only confirmed with three anti-AT1R antibodies directed against C-terminal epitopes of the receptor (anti-AT1R-C18, anti-AT1R-G3, and anti-AT1R-(306) sc-579 (Supplementary Fig. 7 and 8), with the former two antibodies giving signals of tram-track-like appearance in liver cryosections. An additional nucleus-associated AT1R appearance was suggested by two anti-AT1R antibodies, i.e. anti-AT1R-(306) sc-579 ( Supplementary Fig. 8a) and anti-AT1R-NBP1-70997 ( Supplementary Fig. 8b). In all cases, increasing antibody concentrations or prolonged incubation resulted in higher background (data not shown). Note of worth, the performance of anti-AT1R-G3 depended strongly on the lot number, resulting in a high background.
Taken together, the used antibodies performed optimal in air-dried cryosections, treated with ice-cold acetone and permeabilized with Triton X-100 plus Tween-20. Worth of note, when sections were fixed with PFA, antibodies performed poorly as shown for anti-AT1R-C18 ( Supplementary Fig. 1). This might be a reason why other authors described problems, while trying to detect AT1R (Rateri et al. 2011). On the other hand, acetone-treated cryosections are often successfully employed for localization studies in liver and gall bladder (Keon et al. 1996;Anderson et al. 1989;Aust et al. 2004). In our investigations, anti-AT1R-C18 was the superior antibody in both in situ and in vitro studies and was therefore employed when we set out to determine the localization of AT1R.
Identification of AT1R-expressing cells. Histological structures and cell types were identified by features of the DAPI-stained nuclei, such as shape, distribution pattern, and their appearance to luminal space. Cholangiocytes, GBEC, and vascular endothelial cells were further identified by specific antibodies, i.e. anti-CK-19 antibody and anti-CD31 antibody, respectively.
Blocking Peptide. Pre-adsorption of anti-AT1R-C18 with a commercial blocking peptide (1:1, w/w) caused almost complete signal loss in human and porcine hepatocytes ( Supplementary   Fig. 11) and in hhAT1R-expressing HEK293-EBNA cells (Supplementary Fig. 12a). Signal S9 loss was also observed when hhAT1R-expressing HEK293-EBNA cells were incubated with identically pre-adsorbed anti-AT1R-G3 antibody ( Supplementary Fig. 12b). Determination of the blocking peptide sequence by MALDI-TOF (m/z) fingerprint analysis resulted in the corepeptide KYIPPKAKSHS (Supplementary Fig. 13a). This highly conserved polypeptide is located within the last 50 amino acids of the carboxyl terminus of AT1R and is 100% identical with the human and porcine AT1R sequence ( Supplementary Fig. 13b). This led to the conclusion that both the anti-AT1R-C18 and the anti-AT1R-G3 antibodies are directed against the C-terminus of AT1R and that the antibodies detect an AT1R-specific motif.
Detection of heterologous human AT1R protein by Western blotting. Based on the anti-AT1R antibody validation in situ, we employed anti-AT1R-C18 and anti-AT1R-G3 antibodies for detection of AT1R by Western blotting. The specificity of anti-AT1R antibodies was determined in electrophoretically separated total cell lysates from hhAT1R-expressing HEK293-EBNA (T) and empty vector-transfected control (C) cells by Western blotting ( Supplementary Fig. 14a, 14b, left). The used anti-AT1R-C18 and anti-AT1R-G3 antibodies detected unambiguous bands at the expected molecular mass of hhAT1R monomers (42kDa).
The prominent band at a molecular mass approximately double that of the hhAT1R monomer (84kDa), which has been observed before by Barki-Harrington et al. (Barki-Harrington et al. 2003) (Fig. 5a in that work, lower immunoblot, lane 8) suggests the presence of a AT1R homodimer. Homodimerization of heterologous AT1R has previously been shown (Hansen et al. 2004;Hansen et al. 2009;Young et al. 2017). Increased protein load was associated with a signal increase of these bands. Faint bands on the level of AT1R in control lysates suggested the presence of endogenous receptor protein as confirmed by RT-PCR, data not shown. Fig. 14a and 14b, right) produced faint bands at about 60-65kDa. S10 AT1R is well-known as integral plasma membrane receptor. We subjected transfected HEK293-EBNA cells to subcellular fractioning and enriched hhAT1R. The enriched plasma membranes were analyzed by Western blotting and incubated with anti-AT1R-C18 and anti-AT1R-G3 antibodies as well as with the anti-Strep-tag® II antibody (Supplementary Fig. 14c).

Secondary antibody controls (Supplementary
Here, all primary antibodies also detected protein bands corresponding to AT1R monomers and potential homodimers. The additionally performed secondary antibody controls ( Supplementary Fig. 14c) did not result in protein bands, as found for total cell lysates. The monoclonal anti-AT1R-G3 antibody detected an additional weak band of unknown nature in hhAT1R-expressing HEK293-EBNA cells at a molecular mass of about 45kDa.
Both antibodies anti-AT1R-C18 and anti-AT1R-G3 were able to detect hhAT1R in cell lysates and in membrane-enriched fractions. Again, these results were backed up by an anti-Strep-tag® II antibody (Supplementary Fig. 14c). However, AT1R was only detected when a minimum of 30µg total protein was loaded. The loaded target protein concentration and the species-specific primary amino acid sequence determine the recognition by an antibody. The main differences of our study to previous reports were both the protein amount loaded (Herrera et al. 2013a) and the investigated species (Herrera et al. 2013a;Rateri et al. 2011). In these reports, low protein amounts from total tissue or cell lysates were loaded. In addition, these groups aimed to detect rodent AT1R whereas we investigated the human AT1R. Our results are in line with those of Sharma et al. (Sharma et al. 2012) who successfully detected hhAT1R loading 15µg total protein/slot. In our hands, anti-AT1R-antibodies failed to detect AT1R in Western blots of human and porcine liver lysates, even when 200µg protein was loaded. A mass spectrometric analysis of SDS-PAGE-separated lysates (50µg protein) did not reveal the presence of AT1R either, which is most likely caused by the low amount of AT1R protein in the sample. Our results indicated S11 that although AT1R is an abundant receptor in plasma membranes, its amount in the lysates is so low, that it did not meet the antibodies' detection limits. S12      Fig. 1 AT1R-recognition by anti-AT1R-C18 antibody in differently pretreated human liver cryosections (a) Specimens in the top row were air-dried, only. In the row below, the specimens were additionally treated with acetone. In the third row, acetone was replaced by methanol and in S26 row four by an acetone/methanol mixture (1:1). The last two rows show results obtained with 2% and 4% of PFA, respectively. The tram-track-like pattern of bile canaliculi is only visible when specimens were either air-dried or acetone treated. In (b), analogously treated specimens incubated with secondary antibody only. Confocal microscope: Zeiss LSM880. Scale bars: 10 µm (a, b).